A Novel Role for Protein Phosphatase 2A in Receptor-mediated Regulation of the Cardiac Sarcolemmal Na + /H + Exchanger NHE1 *

G q protein-coupled receptor stimulation increases sarcolemmal Na + /H + exchanger (NHE1) activity in cardiac myocytes by an ERK/RSK-dependent mechanism, most likely via RSK-mediated phosphorylation of the NHE1 regulatory domain. Adenosine A 1 receptor stimulation inhibits this response through a G i protein-mediated pathway, but the distal inhibitory signaling mechanisms are unknown. In cultured adult rat ventricular myocytes (ARVM), the A 1 receptor agonist cyclopentyladenosine (CPA) inhibited the increase in NHE1 phosphorylation induced by the α 1 -adrenoreceptor agonist phenylephrine, without affecting activation of the ERK/RSK pathway. CPA also induced significant accumulation of the catalytic subunit of type 2A protein phosphatase (PP2A c ) in the particulate fraction, which contained the cellular NHE1 complement; this effect was abolished by pretreatment with pertussis toxin to inactivate G i proteins. Confocal immunofluorescence microscopic imaging of CPA-treated ARVM revealed significant co-localization of PP2A c and NHE1, in intercalated disc regions. In an in vitro assay, purified PP2A c dephosphorylated a GST-NHE1 fusion protein containing aa 625-747 of the NHE1 regulatory domain, which had been pre-phosphorylated by recombinant RSK; such dephosphorylation was inhibited by the PP2A-selective phosphatase inhibitor endothall. In intact ARVM, the ability of CPA to attenuate the phenylephrine-induced increase in NHE1 phosphorylation and activity was lost in the presence of endothall. These studies reveal a novel role for the PP2A holoenzyme in adenosine A 1 receptor-mediated regulation of NHE1 activity in ARVM, the mechanism of which appears to involve G i protein-mediated translocation of PP2A c and NHE1 dephosphorylation.